Gamma-Aminobutyric acid (GABA) is an inhibitory transmitter at the crustacean neuromuscular junction and it probably also is the major inhibitory transmitter in the vertebrate central nervous system. The level of GABA is believed to be governed by glutamate decarboxylase (GAD), the enzyme which synthesizes GABA from glutamate. This project is designed to study and compare the properties of GAD, isolated and purified both from neurons (GAD I) and glia (GAD II), and to delineate the role of these enzymes in the regulation of GABA as a neurotransmitter. More specifically, I plan to perform the following studies: 1) Purify GAD I and GAD II from the neurons and the glia of calf brain by methods similar to those I had developed for the successful purification of GAD I from the mouse brain; 2) Perform comparative studies of GAD I from mouse and calf brain and of GAD I and GAD II from calf brain. These will include: (a) kinetic studies to determine the enzymic properties such as specificity and affinity for substrate and coenzyme; pH dependence, etc., and to elucidate the possible regulatory mechanism with special emphasis on the effect of metabolites and ions; (b) physical and chemical characterization of the native enzymes and their subunits--namely, the hydrodynamic and optical properties--amino acid composition and analysis of functional groups at the active site with a view to determining the mechanism of decarboxylation; (c) subunit studies which include the elucidation of number of subunits and their identity or non-identity; function of the subunits, e.g., catalytic or regulatory role; hybridization or reconstitution of subunits; (d) immunochemical and ultrastructural studies with a view to determining species-specificities and cell- specificities of GAD from different sources and to locate precisely the cellular site of this enzyme by immunohistochemical probes. The principal investigator has experience with most of the studies mentioned above. The preliminary results which include the purification, characterization, subunit, kinetic and immunochemical studies of GAD I from mouse brain have shown the feasibility of the proposed project.